Journal: The Journal of Biological Chemistry
Article Title: Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells
doi: 10.1016/j.jbc.2022.101747
Figure Lengend Snippet: p38α MAPK inhibition upregulates IL1B-induced TLR2 expression and R1+R2-reporter activity. A , A549 cells were either not treated or pretreated with BIRB796 at various concentrations for 30 min prior to stimulation with IL1B (1 ng/ml). The cells were harvested at 30 min for Western blot analysis and 6 h for qPCR analysis. Blots representative of N independent experiments for phospho-MK2 (P-MK2), total MK2 (MK2), and GAPDH are shown. Densitometric data for P-MK2 and TLR2 mRNA, each normalized to GAPDH, were plotted as fold relative to untreated. B , pHBECs grown in submersion culture were either not stimulated or pretreated with BIRB796 (100 nM) or VX745 (300 nM) for 30 min, followed by addition of IL1B. The cells were harvested for qPCR analysis at 2 and 6 h. U pper panel , TLR2 mRNA was normalized to GAPDH and ( lower panel ) TLR2 unRNA was normalized to U6. In each case, data from N independent experiments were expressed as fold relative to untreated control. C , A549 cells were incubated with concentrations of p38α siRNAs ( si5 , si7 , and si12 ) as indicated, prior to addition of IL1B. The cells were harvested after 30 min and 8 h for western blot analysis. Blots representative of at least four independent experiments are shown. D , A549 cells were incubated with either control siRNA ( siC ), p38α siRNAs ( si5 , si7 , and si12 ), or p38β siRNAs ( si4 , si6 , and si7 ), each at 1 nM, followed by treatment with IL1B. The cells were harvested at 6 h for qPCR analysis. TLR2 mRNA data from N independent experiments was normalized to GAPDH and expressed as fold of untreated. A549 cells stably harboring the R1+R2-luc reporter were either ( E ) incubated with pools of the p38α or p38β siRNAs used in C and D (1 nM) or ( F ) pretreated with BIRB796 (100 nM) or VX745 (300 nM) for 30 min, followed by treatment with IL1B. The cells were harvested for luciferase assay at indicated times. Luciferase activity from N independent experiments was expressed as fold relative to untreated. In B , D , E , and F , significance relative to IL1B was tested by ANOVA with a Dunnett’s post-hoc test. In F , ∗ and $ represent significance of IL1B+BIRB796 and IL1B+VX745, respectively, relative to IL1B. G , A549 cells were either not treated or pretreated with BIRB796 (100 nM) for 30 min, followed by addition of IL1B and/or dexamethasone ( Dex ; 1 μM). The cells were harvested at the indicated times, and ChIP-qPCR for p65 was performed. Data from N independent experiments were plotted as log 2 fold enrichment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. ChIP, chromatin immunoprecipitation MAPK, mitogen-activated protein kinase; pHBEC, primary human bronchial epithelial cell; qPCR, quantitative PCR; TLR, toll-like receptor.
Article Snippet: Budesonide (gift from AstraZeneca), dexamethasone (Sigma-Aldrich), SB203580 (Calbiochem), JNK-IN-VIII (Calbiochem), U0126 (Calbiochem), BIRB796 (Tocris), and VX745 (Tocris) were dissolved in dimethyl sulfoxide as stocks of 10 μM.
Techniques: Inhibition, Expressing, Activity Assay, Western Blot, Control, Incubation, Stable Transfection, Luciferase, ChIP-qPCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction
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